[1] Phospholipase C Enzyme(s)
Conventionally, it is known that animals and microorganisms produce phospholipase C enzyme(s). The major animal-derived enzymes are phosphatidylinositol-selective phospholipase C enzymes. Among microorganism-derived enzymes, phospholipase C enzymes derived from bacteria, actinomycetes, yeast and fungi are known. The phospholipase C enzymes produced by bacteria, actinomycetes and yeast are almost phosphatidylinositol- or glycerophosphorylcholine-selective enzymes.
Known bacteria-derived phospholipase C enzymes include, for example, phospholipase C enzymes produced by Pseudomonas schuylkilliensis (See, for example, Patent Document 1: Japanese Patent Application Kokai Publication No. S50-1017183/1975), Burkholderia pseudomallei (See, for example, Nonpatent Document 1: Korbsrisate S. et al., Journal of Clinical Microbiology, 1999, Vol. 37, p. 3742-3745), Bacillus cereus (See, for example, Nonpatent Document 2: Tan C. et al., Protein Expression and Purification, 1997, Vol. 10, p 365-372), Staphylococcus aureus (See, for example, Nonpatent Document 3: Daugherty S. et al., Infection and Immunity, 1993, Vol. 61, p 5078-5089) and Clostridium perfringens (See, for example, Nonpatent Document 4: Titball R. et al., Infection and Immunity, 1989, Vol. 57, p 367-376).
Known actinomycetes-derived phospholipase C enzymes include, for example, phospholipase C enzymes produced by Streptomyces hachijyoensis (See, for example, Patent Document 2: Japanese Patent Application Kokai Publication No. S49-55893/1974).
Known yeast-derived phospholipase C enzymes include, for example, phospholipase C enzymes produced by Candida albicans (See, for example, Nonpatent Document 5: Andaluz E. et al., Yeast, 2001, Vol. 18, p 711-721) and Saccharomyces cerevisiae (See, for example, Nonpatent Document 6: Payne W. et al., Molecular and Cellular Biology, 1993, Vol. 13, p 4351-4364).
Conventionally, two fungi-derived phospholipase C enzymes are known. One is the phospholipase C produced by Aspergillus niger (See, for example, Patent Document 3: Japanese Patent Application Kokai Publication No. 2000-166543), and the other is one produced by Aspergillus saitoi (See, for example, Nonpatent Document 7: Matsuoka S. et al., Biotechnology and Applied Biochemistry, 1987, Vol. 9, p 401-409).
[2] Lecithin
Lecithin is a representative glycerophospholipid widely distributed among animals, plants and fungi. Glycerophospholipid is a compound having a phosphoryl base covalently bonding to the 3 position of 1,2-diacylglycerol. Choline, ethanolamine, serine, inositol, glycerol, etc. are included as bases, and the composition rate is different depending on sources. The word, lecithin, shall be used as a general idea included in glycerophospholipid.
Lecithin has a surfactant action, antioxidant action, physiological action, etc., and is used for foods, feeds, medicaments, etc. In food industry, the unartificial lecithins, typified by egg yolk lecithin, soybean lecithin, etc., are used as food additives, mainly to modify food properties as an emulsifier and the like, and is supplied abundantly.
[3] Enzyme Treatment of Lecithin
Partially hydrolyzing lecithin enzymatically to provide new properties has also been investigated. Enzymes used for this include phospholipases and phospholipases A, B, C and D are known. Phospholipase A hydrolyzes a fatty acid moiety selectively at the 1 or 2 position of glycerophospholipid. Phospholipase B hydrolyzes glycerophospholipid nonselectively; phospholipase C hydrolyzes glycerophospholipid into diacylglycerol and a phosphoryl base; and phospholipase D hydrolyzes glycerophospholipid into phosphatidic acid and a base.
In the field of food industry, phospholipase A is used comparatively most widely at present. Lecithin is poorly soluble in water and acting phospholipase A on lecithin results in hydrolyzing the acyl group partially to generate water-soluble lysolecithin. When lysolecithin is used as a food additive, the physical properties of the obtained foods may differ from those of foods conventionally obtained using lecithin.
[4] Sphingophospholipid
Sphingophospholipid is composed of phospholipid with glycerophospholipid.
Typical sphingophospholipid is sphingomyelin, a compound in which a choline phosphate is phosphodiester-bonding to the primary alcohol of ceramide. Sphingomyelin is contained in various internal organs of animals. Since sphingomyelin is contained also in breast milk, it may be blended into the powdered infant milk.
Phospholipase C enzyme(s) acts on sphingomyelin to remove a choline phosphate and generate ceramide. Ceramide is widely used in cosmetics as a moisturizing agent. Besides, it's been reported that atopic dermatitis is cause by ceramide deficiency.
[5] Use of Phospholipase C Enzyme(s) in Food
As indicated in the following figure, use of phospholipase C enzyme(s) can produce diacylglycerol from lecithin in the presence of water.
(wherein each of R1 and R2 represents an alkyl group, and R3 represents a group such as choline, ethanolamine, glycerol or inositol.)
By carrying out such reaction in food materials, it may be possible to supply products having different properties essentially from those obtained by acting phospholipase A.
The phospholipase C enzyme(s) used herein is required to hydrolyze various glycerophospholipids. For example, soybean lecithin is known to contain mainly phosphatidylcholine and phosphatidylethanolamine as well as phosphatidylglycerol or phosphatidylinositol, etc. Egg yolk lecithin contains mainly phosphatidylcholine and phosphatidylethanolamine. Therefore, it is desirable that the phospholipase C enzyme(s) used herein hydrolyzes these indiscriminately.
However, a large amount of phosphate in addition to phospholipid are contained in foods, and it is not preferable to hydrolyze them to liberate phosphoric acid, since this further deteriorates the properties of foods. Therefore, phospholipase C enzyme(s) that does not hydrolyze phosphate esters except for phospholipid, is desirable; and for example, phospholipase C enzyme(s), which has no enzymatic activity against phosphate esters such as glycerophosphorylcholine that are similar to phospholipid but do not have lipid moieties, is desirable.
From the same viewpoints, it is desirable that the phospholipase C enzyme(s) used is a protein not having phosphatase activity. That is, the phospholipase C enzyme(s) having no degradation activity of p-nitrophenylphosphate, a substrate of phosphatase, is desirable.
In food industry, it is preferable to carry out various treatments in from a neutral to weak acidic range to prevent deterioration of foods; it is desirable that an enzyme preparation containing phospholipase, etc., has high activity in this pH range.
[6] Use of Phospholipase C Enzyme(s) in Food Industry
The intended purposes of phospholipase C enzyme(s) includes, for example, mitigation of the surface aging and white spots like pear skin during baking frozen bread dough and improvement of purification process of edible oil.
Lecithin is a substance that should be removed, since it causes coloring or deterioration of flavor during manufacturing edible oil from soybean, rapeseed, etc. For this purpose, a method for removing lecithin by partially hydrolyzing lecithin using phospholipase A to lead to aqueous lysolecithin, has been conventionally investigated.
However, here, lecithin can be converted to diacylglycerol using phospholipase C enzyme(s), which is one component of the oil along with triacylglycerol. That is, the improving effect on yields can be expected in the manufacturing process of edible oil.
The phospholipase C enzymes used herein are required to hydrolyze various phospholipids. For example, the above various phospholipids are contained in soybean oils. Similarly, various phospholipids are contained also in cotton seed oils or rapeseed oils. It is desirable that the phospholipase C enzymes used herein hydrolyze these indiscriminately.
Since, in oil mills industries, impurities except for oils are removed under an acidic heating condition, use of an enzyme preparation of which activity is high in an acidic range and which has some degree of heat stability, is desired. Since citric acid may be used to acidify raw oils to be treated, the activity in the presence of citric acid and some degree of heat stability are required for the phospholipase C enzymes used herein.
[7] Problems of Known Phospholipase C Enzymes
The phospholipase C enzymes, produced by animals, bacteria, actinomycetes or yeast, are mainly phosphatidylinositol- or phosphatidylcholine-selective and therefore they are not suitable for use in the food industries in which degradation of various substrates is required. In addition, the enzyme preparation originated from animals cannot be accepted in some countries and areas for religious reasons, and there is also a problem of a wide usability. Furthermore, most bacteria that produce phospholipase C enzymes are pathogenic and therefore there are safety problems.
The phospholipase C enzymes derived from filamentous fungi and known conventionally, have a property to hydrolyze various phospholipids. Besides, any filamentous fungi, conventionally used for the production of phospholipase C enzymes, are characterized in having the actual results in the production of edible enzymes. The enzymes derived from both species of Aspergillus niger and Aspergillus saitoi have extremely similar properties regarding temperature or pH and the same molecular weight. Both enzymes are characterized in having high activity in an acidic range and no activity at around a neutral range. Therefore, these enzymes may be able to be used in oil mills industries where these enzymes may be used in an acidic range. However, properties in citric acid have never been described, and therefore it is unclear whether they can actually be used in oil mills industries (see Patent Document 3 and Nonpatent Document 7). In addition, the activity at pH 6 is near zero and it is difficult to use these enzymes in the food industries where enzyme reactions are often carried out at around neutral pH (see Patent Document 3).
Furthermore, it has been described that the phospholipase C enzymes produced by Aspergillus niger have an extremely high degradation activity of phosphatidic acid, a substrate of phosphatase (see Patent Document 3). Therefore, these enzymes are presumed to be proteins that also have a phosphatase activity, since they have an ability to hydrolyze a phosphoric acid monoester. Furthermore, it has been described that the phospholipase C enzymes produced by Aspergillus saitoi have an extremely high degradation activity of 2-hexadecanoylamino-4-nitrophenyl phosphorylcholine (see Nonpatent Document 7). Therefore, these enzymes are presumed to be proteins additionally to have an ability to hydrolyze any phosphodiesters except for phospholipid. Therefore, it is possible for the properties of foods processed using these enzymes to be changed unexpectedly.
Thus, the phospholipase C enzymes known conventionally do not have sufficient properties as enzymes, or have safety problems, and so forth, and thus no enzyme preparations containing phospholipase C enzymes have been in the market until now. Enzyme preparations having the desirable properties include ones being derived from microorganisms that already have the actual production results as enzyme preparations for foods, having an ability to hydrolyze various phospholipids efficiently both in an acidic range and at around neutral range, having the activity also in a citrate buffer solution to be usable also in oil mills industries, and having some degree of heat stability. Furthermore, the phospholipase C enzymes having a property not to hydrolyze any phosphate esters except for a phospholipid, typified by glycerophosphorylcholine, are included.